Binding assays are a well-established technique for detecting and quantifying analytes in samples. They are particularly useful for detecting and/or measuring substances in biological samples as an aid to disease diagnosis and prognosis, and for predicting a patient's response to therapy. Often they take the form of immunoassays in various formats in which the analyte-binding reagent is an antibody or functional fragment thereof.
The majority of such assays are based on the 2-site or “sandwich” format, which is very useful for analytes that can bind to 2 or more antibodies or receptors simultaneously, but are unsuitable for haptens (which, because of their size, can often only bind to one antibody at a time). For hapten assays, it is usually necessary to utilise saturation-type assays which are typically used for antigen-antibody assays. These are assays whereby sample analyte competes with a fixed quantity of reagent analyte (or analyte analogue, which has been chemically modified such that it is, for example, detectable or immobilised) for a limited number of binding sites on an analyte-binding reagent (e.g. antibody). In saturation assays, the total number of analyte and analyte-analogue molecules is greater than the total number of binding sites on the analyte binding reagent.
These assays can be direct competition format (where sample analyte, analogue and analyte-binding reagent react simultaneously) or indirect competition format (whereby the sample analyte and analyte-binding reagent are allowed to interact together first, and then reacted with the analyte analogue). This latter format is also known as a sequential or back-titre assay. Competition assays generally utilize an analyte analogue labelled with a detectable signal moiety and an unlabelled antibody (frequently attached to a solid phase). The closely-related 1-site immunometric format generally utilizes an antibody labelled with a detectable moiety and an analyte analogue immobilized onto a solid-phase.
The amount of analyte analogue that becomes bound to the analyte binding reagent will thus be inversely proportional to the amount of analyte in sample and by detecting the amount of bound analyte analogue (for competition assays) or bound analyte binding reagent (for 1-site immunometric assays) the presence and/or amount of analyte in a sample can be determined. Detection of a decrease in signal is more difficult than an increase in signal from a negative background, and frequently results in a loss of sensitivity, especially if detection is visual. Measurement of the free or unbound signal overcomes this, but usually the free fraction is present in a larger volume and is diffuse, again resulting in a loss of sensitivity.
There is a growing need for assays to be performed closer to the patient, primarily to shorten the time taken to provide results. Such assays are known as Point-of-Care assays, and typically need to be robust and simple to perform since they are carried out in a non-laboratory setting, frequently by non-skilled staff. Ideally, they should be fully self-contained and require no ancillary equipment (with the possible exception of a reader). Point-of-Care assays need similar sensitivity to laboratory-based assays if they are to have any clinical use. However, conventional immunoassays often comprise complex protocols and detection systems, meaning that they are often unsuitable for point-of-care type use.
Specific Point-of-Care assays have been developed. The most common are lateral flow assays. Often, these are based on a labelled mobile component (e.g. coloured particle-labelled antibody), an immobilised component (e.g. antibody stripe or dot) and a membrane through which sample is caused to move by capillary action. In the presence of analyte, a “sandwich” is formed at the immobilised antibody capture zone, leading to development of a coloured line or dot. Conventional lateral flow assays are exemplified by, for example, U.S. Pat. No. 5,656,503 (Unilever Patent Holdings B.V). These assays specify an immobilised antibody capture zone, albeit in a lateral flow format as opposed to the radial format taught by Geigel et al (1982, Clin Chem 28: 1894).
The basic lateral flow format has been modified to enable competition-type assays to be performed. Thus modified lateral flow assays may be based on a labelled mobile component (e.g. coloured particle-labelled antibody), an immobilised component (e.g. an analyte analogue in the form of a stripe or dot) and a membrane through which sample is caused to move by capillary action. In the absence of analyte, the antibody-labelled particle will bind to the immobilised analyte analogue, leading to development of a coloured line or dot, in the presence of analyte, the binding sites on the antibody will be occupied such that the binding of the particle-labelled antibody is reduced or abolished, with a concomitant reduction or abolition of colour. Such an assay is taught, for example, by Biosite (U.S. Pat. No. 5,143,852). Detection of the decrease in colour, however, can be problematical as described above.
Lateral flow assays offer many advantages, including speed, convenience, and relatively low cost. However, they have several drawbacks. The capture component (e.g. antibody) is generally immobilised by adsorption onto the membrane, so variations in membrane and/or antibody batch can lead to variations in the amount of antibody immobilised. Further, some of the antibodies may be only loosely bound and can become mobile when the fluid front passes, leading to loss of signal. Also, since one antibody is immobilised, the only time for it to react with the analyte is as the sample flows past, so sensitivity can be reduced due to the short incubation time. It is also necessary to produce specific coated membranes for each analyte, thus increasing manufacturing costs.
Attempts have been made to address these disadvantages by avoiding the use of an immobilised capture antibody. For example, EP 297292 (Miles), EP 310872 (Hygeia Sciences), and EP 0962771 (Mizuho) describe systems involving a membrane with a trapping zone in conjunction with 2 antibody-coated particles, one unlabelled but large such that it is trapped by the zone, the other small and labelled which can pass through the zone. In the presence of analyte, the small beads become bound to the trapped large beads, leading to formation of a coloured line. Although these methods avoid the use of a pre-immobilised capture antibody, they require two populations of antibody-coated particles in addition to a trapping zone. Frequently such particles are hydrophobic in nature, and thus can be caused to aggregate in a non-specific manner in the presence of biological fluids.
Others have attempted a simpler format, whereby antibody-coated particles capable of free movement through a membrane are caused to agglutinate in the presence of analyte such that their movement is halted. Such agglutination-based immunoassays are known in the art, and rely upon agglutination of particles to which an antigen or antibody is bound to indicate the presence of the corresponding antibody or antigen in a sample. In one of the simpler forms of an agglutination assay, antibodies to a particular analyte are bound to a bead or other visible material.
In particular, U.S. Pat. No. 4,666,863 (Amersham) discloses a method for separating free and bound label by chromatographic means. In one variant, they teach separation of agglutinated and non-agglutinated antibody-coated coloured particles using flow along a membrane. Prior to separation, the reaction mixture is reacted with a cross-linking agent to stabilise the agglutinate. EP 293779 (Daiichi) also discloses a coloured latex agglutination reaction, where agglutinated and non-agglutinated particles are separated by a capillary which allows non-agglutinated latex through but traps the aggregates. EP 280559 (Kodak) describes an assay for multivalent analytes whereby in the absence of analyte label can pass through a filter, but in the presence of analyte an agglutinate is formed which is trapped. U.S. Pat. No. 6,472,226 (Genosis) describes a lateral flow assay without immobilised antibody for very large analytes. They describe a two-zone system, one having large pores and one having small pores, such that analyte can pass through the large pores but becomes trapped on reaching the zone of small pores. This is used in conjunction with a small label (e.g. gold sol to which antibody is attached) which can pass through both zones. In the presence of analyte, a fraction of the antibody-labelled gold sol becomes bound to the analyte and becomes trapped at the small pore zone.
In the main, these agglutination-based assays are restricted to the detection of large analytes with multiple epitopes which enable the formation of large, stable agglutinates. Their effectiveness with smaller analytes having fewer epitopes, or where only a limited number of available epitopes are being used, can be compromised as the reduced number of binding events may result in a weakened aggregate and loss of sensitivity.
An alternative is the so-called membrane agglutination system (Platform Diagnostics, GB patent application 0523124.6), which is based on immunoagglutination within a capillary membrane such that in the presence of analyte, an agglutinate containing a labelled signal moiety is formed which becomes trapped and generates a detectable signal. The technology is an improvement over earlier systems such as that developed by Amersham International plc, but there remain some aspects that could be improved further. First, the “line” formed by the trapped agglutinate is somewhat broad. Whilst in itself not a problem, it differs slightly from that observed in the more common conventional lateral flow assays. This is likely a result of the system having to accommodate the trapping of particle agglutinates of varying sizes, with the smaller ones probably migrating further into the trapping zone. Secondly, any aggregates present in the labelled particle preparation caused by non-analyte mediated interactions may cause a background signal and false-positive results (the same applies to conventional lateral flow assays). Although this can be overcome by suitable conditions for preparing and applying the particles, it would be advantageous to have a system which avoided the need for this, simplifying manufacture and making a more robust system.
In the main, however, prior art membrane agglutination assays are reliant on the sandwich principle to promote an agglutination reaction. Competition or agglutination-inhibition assays (e.g. haemagglutination inhibition assays for the early pregnancy tests) are known in the art, but have mainly been restricted to assays performed on a slide or in a reaction well and where the agglutination is detected visually by observing a change in the pattern of the particles. Since there is no separation of agglutinated and non-agglutinated particles there is no requirement for the formation of strong, stable agglutinates as they are not exposed to strong shear forces, such as capillary forces.
Attempts have been made to perform competition membrane agglutination assays that detect the free or non-agglutinated fraction of particles to avoid the need to detect a reduction in signal at the separation zone. Angenics Inc (U.S. Pat. No. 4,459,361) describe a particle immunoagglutination system whereby agglutinated and non-agglutinated particles are separated by a filter, and one can measure either an increase in the filtrate (non-agglutinated) or decrease in the retentate (agglutinated) fractions. Akers (EP 556202) describes a similar system in which a test mixture is formed by contacting the sample with coloured particles having analyte-specific receptors on their surface. The test mixture is passed through a filter having pores which are larger than the coloured particles but smaller than the particle-analyte aggregates, thus causing trapping of the aggregates. Presence of analyte in the mixture is determined by checking the colour of the filtrate.
An important further problem with the majority of competition assays of the prior art, including many of the competition membrane agglutination systems described, is that the end point is a reduction in signal of the bound label. Whilst this can be accurately assessed using instrumentation to quantify the signal, it is more difficult to achieve with an assay relying on visual detection (e.g. the majority of Point-of-Care assays), especially at low analyte concentrations where the reduction in signal is small. Assays relying on such signal reduction are therefore not popular for such applications. The membrane agglutination systems that measure the non-agglutinated or free fractions suffer from the diffuse signal, and thus a loss of sensitivity, common to measurements of the free fraction in other assay formats.
A competition membrane agglutination format has been devised utilising antibody-labelled signal particles and analyte- or analyte analogue-coated hubs (or vice versa) which overcomes the drawbacks of the prior art. In the absence of sample analyte, the hub molecules cross-link the labelled particles producing a stable agglutinate which becomes trapped in the membrane (and thus a coloured line). In the presence of sample analyte, some of the antibody sites will be occupied and so the agglutination reaction (and thus signal) reduced or abolished.
Immunoassays are known in which an excess of labelled reagent is detected at a so-called ‘test complete line’, a zone situated at the end of the assay strip comprising immobilised antibody or other binding agent, designed to provide a positive control to confirm the correct flow of solvent and the presence of labelled detection particles. However, such indicators can only be used to reliably report the completion of the test in formats where the labelled reagent is always in excess. They are not, therefore, suitable for saturation assay formats where, in the absence of analyte, most or all of the labelled reagent will be bound in the competition binding step.